The Hox genes are an evolutionarily ancient family of transcription factors critical for proper embryonic development and pattern formation in all animals, including humans. Despite the importance of Hox genes our understanding of the way in which these genes coordinate the expression of large batteries of target genes and thereby control development, is unclear. In particular we do not know the cis-regulatory requirements for Hox gene regulation. We do not know what kind of Hox binding sites a Hox target enhancer requires to be Hox responsive, nor do we know what the topology of these binding sites must be relative to the other regulatory inputs to the enhancer. This proposal is designed to use synthetically modified enhancers to determine the numerical and topological cis-regulatory requirements for target gene regulation by the Hox gene Ultrabithorax. In so doing it should increase our understanding of the way in which the fundamentally important Hox gene family controls embryonic development and may shed light on the general principles of transcriptional regulation, the control of large genetic regulatory networks, and the evolution of genetic regulatory networks over time.